Hepatitis C virus (HCV) is a major cause of liver-specific morbidity and mortality worldwide, resulting in >350,000 deaths annually due to cirrhosis and cancer with high rates of disease among those co-infected with human immunodeficiency virus (HIV) or having a history of injection drug use. microRNA-122 (miR-122), an abundant, liver-specific miRNA is essential for replication of infectious HCV and thus plays a novel role in the pathogenesis of chronic hepatitis C. Recent chimpanzee studies have validated miR-122 silencing as an effective antiviral strategy, yet how miR-122 promotes HCV replication is not well understood. Recent work shows that miR-122 promotes viral translation through direct interactions with a miRNA response element at the 5' end of (+)-strand RNA, while new preliminary data indicate that it also acts to stabilize the RNA. This project will investigate the hypothesis that miR-122, in association with Ago and possibly other cellular proteins, forms a ribonucleoprotein complex (the miRNA-associated stabilization complex, or MASC) at the 5' end of (+)-strand HCV RNA. The MASC complex has several proposed functions: (i) it protects (+)-strand RNA within the cytoplasmic compartment from degradation, thereby increasing RNA available for translation and assembly into membrane-bound replicase complexes; (ii) it may directly enhance the translational activity of HCV RNA; and (iii), it may promote new viral RNA synthesis by facilitating macroassembly of replicase complexes and/or de novo initiation of RNA synthesis. A series of interrelated aims will rigorously test these hypotheses, determine RNA and protein components of the MASC, and further characterize the novel functions of this unique ribonucleoprotein complex. Aim 1 will determine: (a) how miR-122 base-pairs with (+)- strand HCV RNA within the MASC complex; (b) whether RNA stabilization and MASC-increased translation can be functionally uncoupled, and; (c) whether MASC function requires HCV sequences outside the response element. Aim 2 will: (a) combine RNA affinity selection and quantitative proteomics analysis to identify protein components of the MASC complex; (b) validate the presence of these proteins in the MASC complex by IP and assess their role in miR-122 function by RNA interference; (c) develop a cell-free system in which MASC function is recapitulated and the role of host proteins can be further characterized, and; (d) ascertain whether HCV RNA is stabilized by tethering of Ago or other proteins to the 5' RNA. Aim 3 will determine: (a) whether miR-122 regulates macroassembly or stability of replication vesicles by live cell imaging, (b) whether miR-122 is required for negative-strand RNA synthesis, and (c) whether miR-122 facilitates separation of duplexed strands within replicative forms of HCV RNA. Collectively, these studies will provide novel insights into functions of a miRNA that: (a) are important in the pathogenesis of human disease, (b) represent a well-validated but poorly understood therapeutic target, and (c) are unique not only among mammalian viruses but among the Metazoa.